1. Field of the Invention
The present invention relates to an improvement in assays of the type wherein the presence of the analyte is detected by determining a change in refractive index at a solid optical surface, which change is caused by the analyte involving or influencing the binding of a refractive index enhancing species to the optical surface, or the release therefrom, respectively.
One type of method for determining such refractive index changes at an optical surface is based upon surface plasmon resonance, hereinafter SPR. The phenomenon of SPR is well known. In brief, SPR is observed as a dip in intensity of light reflected at a specific angle from the interface between an optically transparent material, e.g. glass, and a thin metal film, usually silver or gold, and depends on among other factors the refractive index of the medium (e.g., a sample solution) close to the metal surface. A change of refractive index at the metal surface, such as by the adsorption or binding of material thereto, will cause a corresponding shift in the angle at which SPR occurs. To couple the light to the interface such that SPR arises, two alternative arrangements are used, either a metallized diffraction grating (Wood's effect), or a metallized glass prism or a prism in optical contact with a metallized glass substrate (Kretschmann effect). For further details on SPR, reference is made to our WO 90/05295. In an SPR-based immunoassay, a ligand may be bound to the metal surface, and the interaction thereof with an analyte of an aqueous sample in contact with the surface is monitored.
Water and diluted aqueous buffers have a refractive index of about 1.33, whereas most proteins have a refractive index in the region of about 1.5 to 1.6. Since the SPR-measurement response is proportional to the change in refractive index caused when, e.g., protein molecules are adsorbed to the surface and displace water therefrom, the refractive index difference between the protein and the buffer solution puts a theoretical limit to the strength of response that may be obtained.
It is understood that SPR-based immunoassays for substances of low molecular weight or occurring in low concentrations, like, for instance, haptens, are problematic due to the very small changes in refractive index caused when the analyte binds to or dissociates from the antibody-coated sensing surface.
2. Description of Related Art
Attempts to obviate this problem in SPR-immunoassays are described in EP-A-276 142 and WO 90/11525 (the former specifically making use of the above mentioned Wood's effect, and the latter of the Kretschmann effect).
Both publications disclose the conjugation of a reagent (for example the analyte or an analyte analogue in a competition assay) with a refractive index increasing species or probe. Such a probe may, for instance, be a molecule or particle having a high refractive index and/or a large size. Possible substances include heavy substances (such as metal ions or higher halogens), highly electronically delocalized species (such as polycyclic aromates or dyes), metal or metal oxide particles (such as titania particles), or high refractive index organic species (such as ferritin). The substance may, alternatively, be one of a lower refractive index than that of the environment close to the sensing surface. The substance may also be an enzyme causing the production of a reaction product which is deposited on the sensing surface and which has a refractive index higher or lower than the material present in the thin layer at the sensing surface.
However, among organic species it is difficult to find molecules having a refractive index higher than about 1.6-1.7. Inorganic species, on the other hand, may have refractive indices of about 2-3, but instead they may be difficult to combine chemically with proteins. The possibilities of significantly enhancing the SPR-signals as proposed by the two cited publications are therefore rather limited, unless, of course, extremely large and thereby impractical probes are used. The problem of insufficient sensitivity for certain kinds of analytes in the mentioned types of assays therefore still remains.